ES2385888T3 - Stable aqueous solution of human erythropoietin that does not contain serum albumin - Google Patents

Abstract

The present invention provides an aqueous formulation of human erythropoietin having the storage stability over a long period without serum albumin, in which the formulation comprises a pharmaceutically effective amount of human erythropoietin; non-ionic surfactant, polyhydric alcohol, neutral amino acid and sugar alcohol as stabilizers; isotonic reagent; and buffering reagent.

Description

Stable aqueous solution of human erythropoietin that does not contain serum albumin.

Field of the Invention

The present invention relates to an aqueous formulation of human erythropoietin that has storage stability for a long period of time without serum albumin. More specifically, the present invention relates to the formulation comprising a pharmaceutically effective amount of human erythropoietin; a non-ionic surfactant, a polyhydric alcohol selected from the group consisting of propylene glycol and glycerol, a neutral amino acid and a sugar alcohol as stabilizers; isotonic reagents; and a buffer reagent, the pH of the buffer reagent ranging between 6.0 and 7.5.

Background of the invention

Erythropoietin (EPO) is a glycoprotein that induces the production of erythrocytes in the bone marrow by stimulating the differentiation of erythroid progenitor cells. EPO consists of 165 amino acids. After in 1977 Mijake purified erythropoietin from human urine, it is currently possible to produce it in large quantities using genetic engineering technologies. It has been proven that erythropoietin is capable of effectively inducing various hematopoiesis in the treatment of anemia resulting from chronic renal failure and in various types of anemia of varied origin, being during certain surgical procedures (Mijake et al., J. Biol. Chem .25, 5558-5564, 1977; Eschbach et al., New Engl. J. Med. 316, 73-78, 1987; Sandford. BK, Blood, 177, 419-434, 1991; WO 85-02610). For this reason, erythropoietin has been used for a long time as a medicine in various indications of diseases. However, like other protein pharmaceuticals, erythropoietin must also be carefully prepared to avoid denaturation caused by loss of stability, in order to use it effectively.

In general, proteins have a short half-life and denaturation occurs easily, for example by aggregation of monomers, precipitation by aggregation and adsorption on the blister walls when exposed to extreme temperatures, a water and air contact surface, at high pressure, physical and mechanical stresses, organic solvents, contamination by microorganisms and the like. Denatured proteins lose their physicochemical properties and their native physiological activity, being this denaturation of proteins generally irreversible. Thus, once denatured, proteins cannot recover their native properties. Especially in the case of proteins such as erythropoietin, which is administered in single doses of only a few micrograms, when they are adsorbed on the bleb wall due to instability, the resulting loss is relatively considerable. In addition, the protein thus adsorbed is easily added via a denaturation process and the administration of the denatured protein causes the appearance of antibodies, such as proteins produced spontaneously, against this denatured protein in the body, so the protein must be administered in an essentially stable way. Therefore, various methods for preventing protein denaturation in aqueous solution have been studied (John Geigert, J. Parenteral Sci. Tech, 43, No. 5, 220-224, 1989; David Wong, Pharm. Tech October, 34-48, 1997; Wei Wang, Int. J. Pharm., 1 85, 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986; Michelle et al., Int. J. Pharm. 120, 179-188,1995).

Some protein formulations solve denaturation with a lyophilization method. However, lyophilized products are inconvenient, since they have to be reconstituted before injection and a high capacity lyophilizer is also required for processing, so a large investment is necessary. Methods for producing protein powder forms are also applied using spray drying techniques. However, this method has the disadvantages that economic efficiency decreases due to poor performance and that exposure to high temperatures can cause protein denaturation during the process.

As an alternative way to solve the limitations of the methods described above, there is a method to improve protein stability by adding stabilizers to an aqueous protein solution. As protein stabilizers, surfactants, serum albumin, polysaccharides, amino acids, macromolecules and salts are known (John Geigert, J. Parenteral Sci. Tech., 43, No. 5, 220-224, 1989; David Wong, Pharm. Tech., October, 34-48, 1997; Wei Wang., Int. J. Pharm., 185, 129-188, 1999). However, suitable stabilizers should be selected according to the physicochemical characteristics of each protein, since, otherwise, for example if stabilizers are used in certain combinations, a competitive reaction or a secondary reaction with negative effects other than provided. In addition, since for each stabilizer there is an appropriate concentration range, much effort and prudence is required to stabilize aqueous proteins (Wei Wang, Int. J. Pharm., 185, 129-188, 1999).

Among protein stabilizers, serum albumin and gelatin, of human or animal origin, are generally used as stabilizers for aqueous protein formulations, proving to be effective. However, serum albumin of human origin poses a risk of viral contamination and bovine serum albumin and gelatin can transmit diseases such as "Transmissible Spongiform Encephalopathies" or cause allergies in some patients. Therefore, in Europe, the use of materials of human and animal origin as pharmaceutical additives is increasingly restricted (EMEA / CPMP / BWP / 450/10 Report from the Expert Workshop on Human TSEs and Medicinal products derived from Human Blood and Plasma (December 1, 2000), CPMP / PS / 201/98 Position Statement on New Variant CJD and Plasma-Derived Medicinal Products (Replaced by CPMP / BWP / 2879/02). Therefore, it is necessary to develop methods for formulating protein formulations stable without serum albumin of human or animal origin and thus solve the problems of existing erythropoietin formulations containing serum albumin.

US Patent 4,879,272 describes the addition of human / bovine serum albumin, lecithin, dextran and cellulose as agents for inhibiting the adhesion of proteins to the blister walls. According to this patent, the recovery performance of erythropoietin is good with 69 � 98% after storage for approximately 2 hours at 20 ° C, compared to a yield of only 16% without such addition, but has the problem that The loss due to adsorption can be considerable.

US 4,806,524 discloses a lyophilized formulation and an aqueous formulation of erythropoietin where polyethylene glycol, protein, saccharides, amino acids, organic salts and inorganic salts are used as erythropoietin stabilizers. According to this patent, after storage of approximately 7 days at 25 ° C, the lyophilized formulation has a high recovery yield of 87 98%, but the aqueous formulation has a low recovery yield of only 60 70 %, so the aqueous formulation is relatively less stable.

In US Patent 4,992,419 an aqueous formulation and a lyophilized formulation of erythropoietin are described where 0.5 ± 5 g / l of non-ionic surfactant are used as anti-absorption agent and 5 � 50 g / l of urea and 5 � 25 g / l of amino acids as stabilizers. However, this patent has the problem that the aqueous formulation has limited stability compared to formulations containing human serum albumin and that the lyophilized formulation requires a reconstitution process to maintain sufficient activity.

In US Patent 5,376,632 an aqueous formulation, a lyophilized formulation and a powder-dried formulation of erythropoietin containing 1 o and cyclodextrins but no other additional pharmaceutical excipient are described. However, formulations where cyclodextrins are used are not practical due to their renal toxicity.

In US Patent 5,661,125 a formulation of erythropoietin containing benzyl alcohol, parabens, phenol and mixtures thereof is disclosed, and an experiment demonstrating its stability compared to a formulation of erythropoietin containing human serum albumin. However, the formulation of this patent has low stability and significant precipitation of erythropoietin even at low temperatures.

In WO 01/87329 A1, aqueous formulations of erythropoietin and a multi-loading inorganic anion in a pharmaceutically acceptable buffer to maintain the pH of the solution in the range between about pH 5.5 and about pH 7.0 are described. This application presents a comparative experiment referring to stability where, after storage of EPO and PEGylated EPO at different temperatures for 6 months, the sialic acid content and the standard bioactivity (%) of each EPO in different formulations were measured. However, since the amount of EPO monomers was not measured, the recovery yield (%) of EPO monomers cannot be determined accurately.

EP 0 909 564 discloses the preparation of an erythropoietin solution containing an amino acid as stabilizers and with excellent long-term storage stability.

WO 00/61169 describes aqueous pharmaceutical formulations of erythropoietin free from human serum blood products, stabilized with amounts of amino acid and a sorbitol mono-9octadecanoate derivative poly (oxo-1,2-ethanedyl).

Therefore, it is desirable to provide a new aqueous formulation that exhibits long-term stability without using protein components derived from animals such as serum albumin.

Summary of the invention

The object of the present invention is to provide an aqueous formulation of erythropoietin that can maintain its biological activity for long periods of time in vivo without using serum albumin of human or animal origin.

Thanks to many experiments and intensive studies, the inventor has found an aqueous formulation of human erythropoietin that prevents adhesion to the blister wall and denaturation of proteins that occurs with storage for long periods, by combining a pharmaceutically effective amount of erythropoietin with specific components such as stabilizers, isotonic reagents and buffer, and carried out the invention.

Detailed Description of the Invention

Thus, the present invention provides an aqueous formulation of human erythropoietin without serum albumin, which comprises a pharmaceutically effective amount of human erythropoietin; a nonionic surfactant, a polyhydric alcohol selected from the group consisting of propylene glycol and glycerol, neutral amino acids, a sugar alcohol as stabilizers; an isotonic reagent; and a buffer reagent, the pH of the buffer reagent ranging between 6.0 and 7.5.

Human erythropoietin that can be used in the aqueous formulation of the present invention includes all types of erythropoietin obtainable by isolation and purification of animal cells by native and / or genetic recombinant methods. The amount of erythropoietin in the aqueous formulation preferably ranges from 100 IU / ml to 120,000 IU / ml.

The aqueous formulation of the present invention contains a non-ionic surfactant to stabilize the formulation, preventing adhesion to the blister walls. The nonionic surfactant decreases the surface tension of the proteins, preventing their adhesion or aggregation on the hydrophobic surfaces. Examples of preferred non-ionic surfactants for use with the present invention include non-ionic surfactants based on polysorbate and based on poloxamer, being able to be used individually or in combination of two or more thereof. Among these, polysorbate-based nonionic surfactants are especially preferred. Examples of such polysorbate-based nonionic surfactants include polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80, and, among these, polysorbate 20 is particularly preferred. Polysorbate 20 inhibits chemical degradation of proteins and decreases or prevents their adhesion at low concentration, since their Critical Micellar Concentration is relatively low. Preferably, high concentrations of the nonionic surfactant are not used in the aqueous formulation, since such concentrations cause interference in the UV spectroscopy and at the isoelectric point during the stability and protein concentration test, which makes it difficult to assess its stability. Therefore, the non-ionic surfactant is incorporated into the aqueous formulation of the present invention in amounts less than 0.01%, particularly preferably between 0.0001 and 0.01% (weight / volume).

The neutral amino acid allows many more water molecules to be present around erythropoietin, which makes it possible to stabilize the outermost hydrophilic amino acids of erythropoietin, thus stabilizing erythropoietin itself (Wang, Int. J. Pharm. 185 (1999) 129 -188). Neutral amino acids are used in the aqueous formulation of the present invention because charged amino acids can facilitate aggregation of erythropoietin by electrostatic interaction. Examples of preferred neutral amino acids to be used in the present invention include glycine, alanine, leucine, isoleucine, etc. Among these, glycine is especially preferred. These neutral amino acids can be used individually or in combination of two or more thereof. However, according to the experiments performed by the present inventors, glycine is more effective when used alone than when used in combination with other amino acids. However, the aqueous formulation of the present invention should not be limited to the use of a neutral amino acid type. The amount of neutral amino acid to be used in the present invention preferably ranges from 0.001 to 2% (w / v). If the amount is less than this range, there may be no effect of increasing stability. On the other hand, if the amount is greater than this range, a high concentration of erythropoietin is not achieved, since the high osmotic pressure influences its solubility.

In the aqueous formulation of the present invention, polyhydric alcohol is used as one of the stabilizers of erythropoietin in solution. Polyhydric alcohols include propylene glycol, glycerol and one or a combination of two or more thereof. Especially, among these, propylene glycol is particularly preferred. Propylene glycol has been widely used in pharmaceutical products to be administered parenterally or non-parenterally as a solvent of hydrophobic materials, extractant and preservative, and is considered non-toxic. In addition, it is used as an emulsifier or as a vehicle in food and cosmetics. Apart from the above, it can also be used as a stabilizer in pharmaceutical products to increase the solubility of phospholipids if they are used as stabilizers of aqueous formulations. Propylene glycol can also be used to increase the stability of aqueous protein formulations and, in general, to a greater extent improve the stability of aqueous formulations when used in combination with other stabilizers in suitable concentrations than when used alone. However, it should be noted that the present inventors have found that, despite the use of propylene glycol, the stability of the aqueous formulation decreases unexpectedly if the types and concentration ranges of other stabilizers used are not properly selected in combination with propylene glycol. The amount of polyhydric alcohol preferably ranges from 0.0001 to 0.1% (w / v). If the amount is less than this range, there may be no effect of increasing stability. On the other hand, if the amount is greater than this interval, problems may occur due to the increase in osmotic pressure.

Sugar alcohol, as one of the stabilizers of the aqueous formulations of the present invention, performs its stabilizing function of erythropoietin when supplied in solution with the non-ionic surfactant, neutral amino acid and polyhydric alcohol as mentioned above. above. Examples of preferred sugar alcohols include mannitol, sorbitol, cyclitol, inositol, etc., which can be used individually or in combinations of two or more thereof. Among them, mannitol is especially preferred. The amount of sugar alcohol preferably ranges from 0.1 to 1.0% (w / v). If the amount is less than this range, there may be no effect of increasing stability. On the other hand, if the amount is greater than this interval, problems may occur due to the increase in osmotic pressure.

Certain stabilizers, for example sugar alcohol and the like, may not be limited to the literal meaning of the term itself, but, in some cases, it is also provided that they perform other functions for the preparation of the aqueous formulation according to the present invention, by example a function as an isotonic reagent.

The isotonic reagent, used as another component of the aqueous formulation of the present invention, serves to maintain osmotic pressure when erythropoietin is administered as a solution to an organism, also having an additional effect of increasing the stability of erythropoietin in solution. Representative examples of isotonic reagents include water soluble inorganic salts, including these salts, for example, sodium chloride, calcium chloride, sodium sulfate, etc. These salts can be used individually or in combinations of two or more thereof. Among them, sodium chloride is especially preferred. The amount of water-soluble inorganic salt preferably ranges from 0.001 to 0.7% (w / v) and can be appropriately adjusted so that the aqueous formulation, which contains various components as described above, is isotonic.

The combination of the stabilizers indicated above with the isotonic reagent, contained in the aqueous formulation, stabilizes the erythropoietin in solution synergistically and not competitively with each other. According to the studies of the present inventors it has been found that, for example, although propylene glycol has a stabilizing effect of erythropoietin in solution to some extent even when used alone, this stabilization effect can be further increased if used in combination with neutral amino acids.

Therefore, if any one of the above-mentioned isotonic stabilizers or reagents is eliminated, there is a surprising decrease in the stability of erythropoietin. This is demonstrated in the Examples and Comparative Examples illustrated below.

In the aqueous formulations of the present invention, the buffer reagent serves to maintain the pH of the solution in order to stabilize the erythropoietin. Examples of preferred buffer reagents include phosphate buffer, citrate buffer and the like. Among these, phosphate buffer is especially preferred. For example, the concentration range of the phosphate that makes up the phosphate buffer reagent is preferably 5 50 mM and the pH range of the solution is about 6.0 7.5, a range of about 6.5 7 being especially preferred ,5.

In the aqueous formulation of the present invention, in addition to the stabilizers, the isotonic reagent and the buffer reagent, any other substances or materials known in the art can also be selectively included, in concentration ranges that do not negatively influence the effects of the invention.

Examples of formulations of the present invention are illustrated in detail below. However, said examples of formulations are only illustrative of the present invention and, consequently, the present invention is not limited to the following examples.

Example 1: Preparation of an aqueous formulation of human erythropoietin -1

An isotonic solution was prepared by adding 0.003% polysorbate 20, 0.1% propylene glycol, 1.5% glycine, 0.1% sodium chloride and 1.0% mannitol to a solution of 10 mM phosphate buffer, and erythropoietin (LG Life Science Co., Ltd.) was added at approximately 4,000 IU / ml. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 1:

Preparation of an aqueous formulation of human erythropoietin without additives

Erythropoietin in an amount of 4,000 IU / ml was added to a 10 mM phosphate buffer solution. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 2:

Preparation of an aqueous formulation of human erythropoietin containing only polysorbate 20

Erythropoietin in an amount of 4,000 IU / ml was added to a 10 mM phosphate buffer solution containing 0.003% polysorbate 20. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these They were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 3: Preparation of an aqueous formulation of human erythropoietin without propylene glycol

An isotonic solution was prepared by adding 0.003% polysorbate 20, 1.5% glycine, 0.1% sodium chloride and 1.0% mannitol to a 10 mM phosphate buffer solution, and adding erythropoietin at approximately 4,000 IU / ml. Then 2 ml aliquots of the solution were carefully transferred

5 prepared in 3 ml glass vials and these were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 4: Preparation of an aqueous formulation of human erythropoietin without glycine

An isotonic solution was prepared by adding 0.003% polysorbate 20, 0.5% propylene glycol, 0.1% sodium chloride and 1.0% mannitol to a 10 mM phosphate buffer solution, and adding erythropoietin at about 4,000 IU / ml. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 5: Preparation of an aqueous formulation of human erythropoietin without sodium chloride

An isotonic solution was prepared by adding 0.003% polysorbate 20, 1.7% glycine, 0.5%

15 propylene glycol and 1.0% mannitol to a 10 mM phosphate buffer solution, and erythropoietin was added at approximately 4,000 IU / ml. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these were sealed and stored at 25 ° C and 37 ° C, respectively.

Comparative Example 6: Preparation of an aqueous formulation of human erythropoietin without mannitol

An isotonic solution was prepared by adding 0.003% polysorbate 20, 0.5% propylene glycol, 1.5% glycine and 0.1% sodium chloride to a 10 mM phosphate buffer solution, and added erythropoietin at approximately 4,000 IU / ml. Then 2 ml aliquots of the prepared solution were carefully transferred to 3 ml glass vials and these were sealed and stored at 37 ° C.

Experimental example 1: 25 Stability of aqueous formulations of human erythropoietin

The relationship between the erythropoietin monomers and dimers of the aqueous formulations of Example 1 and Comparative Examples 1 to 6 was determined using SEC-HPLC after storage for 3 and 5 weeks, respectively. The results are shown in the following Table 1.

Table 1

Yield recovery (%)

Dimer (%)

25ºC

37 ° C  25ºC 37 ° C

3 wks

5 wks  3 wks 5 wks  3 wks 5 wks  3 wks 5 wks

Exp. one

100 98.9 94.2 93.5 0.0 0.0 0.0 0.0

Comm. one

87.1 86.5 78.6 75.6 0.0 0.0 0.0 0.0

Comm. 2

91.1 92.4 83.4 82.6 0.0 0.0 11.1 13.1

Comm. 3

93.4 93.0 85.5 83.7 0.0 0.0 7.2 9.8

Comm. 4

94.1 93.5 87.2 85.1 0.0 0.0 4.3 6.5

Comm. 5

95.2 94.8 90.2 88.3 0.0 0.0 1.35 2.4

Comm. 6

- - 85.1 81.1 - - 8.3 5.5

sem = weeks

As can be seen above in Table 1, Example 1, consisting of an aqueous erythropoietin formulation according to the present invention, showed a recovery yield of more than 92% after storage for 5 weeks at 37 ° C without being detected. No dimer On the other hand, Comparative Example 2, which only contained polysorbate 20 in the formulation, showed high performance compared to Comparative Example 1 free of additives, but dimers were detected. In Comparative Examples 3 to 6, which did not contain any of the components according to the present invention in the formulation, dimers were detected after storage at 37 ° C for 3 weeks, decreasing the recovery yield to approximately 80%. In addition, in Comparative Examples 3 and 4, which did not contain propylene glycol and glycine in the formulation, respectively, the recovery yield was low compared to the formulation of Example 1, with many dimers being detected. The results shown above confirm that propylene glycol and the other stabilizers and isotonic reagent provided by the present invention have synergistic effects when used in combination. It can be seen that, although each of these components added to the formulation of the present invention has a stabilizing effect, the anti-adhesion of erythropoietin in solution and the stability of aqueous erythropoietin can be improved synergistically by the combined effects of every

15 ingredient

Example 2: Preparation of an aqueous formulation of human erythropoietin -2

An isotonic solution was prepared by adding 0.01% polysorbate 20, 0.1% propylene glycol, 0.1% glycine, 0.55% sodium chloride and 1.0% mannitol to a 10 mM phosphate buffer solution, and erythropoietin was added at approximately 4,000 IU / ml. Then 0.5 ml aliquots of the solution were collected

20 prepared in pre-filled syringes of 1 ml (Becton-Dickinson) and these were stored at 40 ° C for 4 weeks.

Comparative Example 7:

Preparation of an aqueous formulation of human erythropoietin without propylene glycol or mannitol

25 To a solution containing 4.38 mg / ml of sodium chloride, 1.16 mg / ml of monosodium phosphate (dihydrate), 2.23 mg / ml of disodium phosphate (dihydrate), 5 mg / ml of glycine and 0.3 mg / ml polysorbate 80 was added erythropoietin at approximately 4,000 IU / ml. Then 0.5 ml aliquots of the solution prepared in 1 ml pre-filled syringes (Becton-Dickinson) were collected and stored at 40 ° C for 4 weeks.

Experimental Example 2:

30 Stability of aqueous formulations of human erythropoietin -2

The purity and recovery performance of erythropoietin from the aqueous formulations of Example 2 and Comparative Example 7 were determined using SEC-HPLC after 0, 1, 3 and 4 weeks, respectively. The results are shown in the following Table 2.

Table 2

Monomer recovery performance (%)

0 weeks

1 week 3 weeks 4 weeks

Exp. 2

100.0 95.8 89.9 92.9

Exp. Comp. 7

100.0 93.9 85.2 86.4

As can be seen above in Table 2, the formulation of the present invention showed a high recovery yield of 92.9% after 4 weeks of incubation, compared with 86.4% of the reference formulation. . Accordingly, the formulation of the present invention improves the stability of erythropoietin in solution.

Comparative Examples 9-13:

40 Preparation of various aqueous formulations of human erythropoietin

The aqueous formulations of Table 3 shown below were prepared, which differed from the aqueous formulations of Example 1 in certain components and were stored at 25 ° C and 37 ° C, respectively, under the same conditions as in Example 1.

Table 3

Composition of the formulations

Exp. com. 9

EPO 4,000 IU / ml in PB, 0.5% PG, 0.003% Tween 20, 0.5 mM sucrose, 1 mg / ml NaCl

Exp. com. 10

EPO 4,000 IU / ml in Tris, 0.5% PG, 0.003% Tween 20, 1.5% Gly, 1 mg / ml NaCl

Exp. com. eleven

EPO 4,000 IU / ml in PB, 0.5% PVP 15K, 0.003% Tween 20, 1.5% Gly, 0.1% NaCl, 1% mannitol

Exp. com. 12

EPO 4,000 IU / ml in PB, 0.5% PG, 0.003% Tween 20, 1.5% Gly, 1 mg / ml NaCl, 0.00001% carboxymethylcellulose

Exp. com. 13

EPO 2,000 IU / ml in PB, 2% PG, 2% glucose, 0.002% lecithin, 0.001% Tween 20

PB: PG phosphate buffer: propylene glycol PVP: polyvinylpyrrolidone

Experimental example 3: Stability of aqueous formulations of human erythropoietin -3

The relationship between the erythropoietin monomers and dimers of the aqueous formulations of Comparative Examples 9-13 was determined using SEC-HPLC at 3 and 5 weeks, respectively. The results are shown in the following Table 4, compared with the results of the aqueous formulations of Example 1.

Table 4

Yield recovery (%)

Dimer (%)

25ºC

37 ° C  25ºC 37 ° C

3 wks

5 wks  3 wks 5 wks  3 wks 5 wks  3 wks 5 wks

Ex. 1

100 98.9 94.2 93.5 0.0 0.0 0.0 0.0

Exp. Com. 9

NA NA 68.1 72.9 NA NA 5.8 12.5

Comm. 10

NA NA 48.2 - * NA NA 21.0 - *

Comm. eleven

** ** ** ** ** ** ** **

Comm. 12

*** *** *** *** *** *** *** ***

Comm. 13

NA NA NA NA 46.6 56.6 59.2 58.9

*: interruption of the test due to precipitation. **: interruption of the test due to precipitation after the preparation of the formulations. ***: interruption of the test by precipitation after the preparation of the formulations. NA: not applicable Comparative example 13: results obtained at 30 ° C and 50 ° C.

As can be seen above in Table 4, replacing some of the components of the aqueous formulations of Example 1 according to the present invention with other compounds did not yield the desired results.

Comparative Example 14:

Preparation of an aqueous formulation of human erythropoietin -3

For the preparation of the aqueous formulations using polyhydric alcohols other than propylene glycol, the aqueous formulations of human erythropoietin were prepared in the same manner as in Example 1, except that 0.025% of PEG 300 (polyethylene glycol, Mn = 300) was used instead of propylene glycol. The prepared solution is

5 introduced into glass vials and these were sealed and stored at 37 ° C.

Comparative experimental example:

Stability of aqueous formulations of human erythropoietin -4

The relationship between the erythropoietin monomers and dimers of the aqueous formulations of Comparative Example 14 was determined using SEC-HPLC at 3 and 5 weeks, respectively. For comparison, the

The aqueous formulation of Comparative Example 3, which did not contain polyhydric alcohol. The results are shown in Table 5.

Table 5

Yield recovery (%)

Dimer (%)

3 weeks

5 weeks  3 weeks 5 weeks

Exp. Comp. 14

100.0 97.2 0.0 0.0

Exp. Comp. 3

85.5 83.7 7.2 9.8

As can be seen above in Table 5, Comparative Example 14, which consists of a formulation

Erythropoietin water in which polyethylene glycol is used as a polyhydric alcohol has a monomer recovery yield of 97%, with no dimer being detected. This result is very different from that of the aqueous formulation of Comparative Example 3 in which no polyhydric alcohol is used.

Example 4: Stability of an aqueous formulation with high concentration of erythropoietin

An isotonic solution was prepared by adding 0.003% polysorbate 20, 0.1% propylene glycol, 1.5%

20 glycine, 1.0% mannitol and 0.1% sodium chloride to a 10 mM phosphate buffer solution, and erythropoietin was added at approximately 12,000 IU / ml. Then 0.5 ml aliquots of the solution prepared in 1 ml pre-filled syringes (Becton-Dickinson) were collected and stored at 5 ° C, 25 ° C and 40 ° C for 3 months, respectively.

Experimental example 5:

25 Stability of aqueous formulations with high concentration of erythropoietin

To confirm the stability of the aqueous formulations with high concentration of erythropoietin, the recovery yield and purity of the formulation prepared in Example 4 were determined by SEC-HPLC and RP-HPLC after storage for 0, 6 and 12 months at 5 ° C, after storage for 2, 4 and 6 months at 25 ° C and after storage for 1, 2 and 3 months at 40 ° C, respectively. The

30 level of physiological activity by administering the formulations to B6D2F1 mice and measuring the increase in reticulocytes. The results are shown in the following Table 6.

Table 6

5 ° C storage

Storage at 25 ° C Storage at 40 ° C

Time (months)

0 6 12 2 4 6 one 2 3

Recovery Performance (%)

100.0 107.8 104.9 100.0 100.0 103.9 100.0 96.1 94.1

Purity (%)

100.0 99.9 99.9 100.0 99.8 99.3 99.8 99.3 99.2

Physiological act (%)

89.7 90.8 NT 90.4 90.8 93.8 105.3 82.8 91.8

As can be seen above in Table 6, the high concentration of erythropoietin in the aqueous formulations showed complete recovery performance, purity and physiological activity during storage of up to 12 months at 5 ° C and also during storage of up to 6 months at 25 ° C. In addition, during recovery of up to 3 months at 40 ° C a recovery performance of a

5 94%, with little decrease in purity and physiological activity. Therefore, it was confirmed that the formulations according to Example 2 were very stable.

Experimental Example 6:

Stability depending on the effects of pH

To a 10 mM phosphate buffer solution was added 0.003% polysorbate 20, 0.5% propylene glycol, a

10 1.5% glycine, 1.0% mannitol and 0.1% sodium chloride, and erythropoietin was added at 4,000 IU / ml. The pH was then adjusted using acetate and sodium hydroxide. The prepared solution was divided into 2 ml aliquots and placed in 3 ml test tubes, and these were sealed and stored at 40 ° C for 3 weeks. The ratio between the erythropoietin monomers and dimers was then measured. The results are shown in the following Table 7.

15 Table 7

pH

Rdto. Recovery (%) Dimer (%)

6.0

94.4 0.0

6.5

95.2 0.0

7.0

94.3 0.0

7.5

90.3 0.0

8.0

81.7 0.0

8.5

69.8 0.0

9.0

55.3 11.8

As can be seen above in Table 7, the monomer recovery yield was greater than 90% even after 3 weeks of storage at 40 ° C in the case of pH values of 6.0 to 7.5. This result demonstrates that the aqueous formulations of erythropoietin according to the present invention, which contain a non-ionic surfactant, propylene glycol, neutral amino acid, sodium chloride and isotonic reagent,

20 are very stable at pH values 6.0-7.5.

Industrial application

As described above, the aqueous formulation of erythropoietin according to the present invention, which contains human erythropoietin and non-ionic surfactant, polyhydric alcohol, neutral amino acid, sugar alcohol as stabilizer, isotonic reagent and buffer reagent, improves the problem. of the

25 decrease in physiological activity due to denaturation during long-term storage in solution. In addition, it has the property of preventing erythropoietin proteins from adhering to the blister walls.

Other examples and uses of the invention will be apparent to those skilled in the art taking into account the description and practice of the invention described herein. The examples are to be considered only as such,

30 the scope of the particular examples of the invention being that of the following claims.

Claims (11)

one.

Aqueous formulation of human erythropoietin without serum albumin, comprising human erythropoietin; a non-ionic surfactant, a polyhydric alcohol selected from the group consisting of propylene glycol and glycerol, a neutral amino acid and a sugar alcohol as stabilizers; an isotonic reagent; and a buffer reagent, the pH of the buffer reagent ranging between 6.0 and 7.5.

2.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that said human erythropoietin is native or recombinant erythropoietin.

3.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the non-ionic surfactant is a non-ionic surfactant based on polysorbate or a non-ionic surfactant based on poloxamer, or a combination thereof; the neutral amino acid is one or more amino acids selected from the group consisting of glycine, alanine, leucine and isoleucine; the sugar alcohol is one or more alcohols selected from the group consisting of mannitol, sorbitol, cyclitol and inositol; the isotonic reagent is one or more reagents selected from the group consisting of sodium chloride, calcium chloride and sodium sulfate; and the buffer reagent is one or more reagents from the group consisting of phosphate buffer and citrate buffer.

Four.

Aqueous formulation of human erythropoietin according to claim 3, characterized in that the non-ionic surfactant is polysorbate 20, the polyhydric alcohol is propylene glycol, the amino acid is glycine, the sugar alcohol is mannitol, the isotonic reagent is sodium chloride and the buffer reagent It is phosphate buffer.

5.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the content of non-ionic surfactant ranges between 0.0001 and 0.01% (w / v).

6.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the content of polyhydric alcohol ranges between 0.001 and 0.1% (w / v).

7.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the neutral amino acid content ranges between 0.001 and 2% (w / v).

8.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the sugar alcohol content ranges between 0.1 and 1.0% (w / v).

9.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the content of said isotonic reagent ranges between 0.001 and 0.7% (w / v).

10.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the salt concentration in the buffer reagent ranges between 1 mM and 50 mM.

eleven.

Aqueous formulation of human erythropoietin according to claim 1, characterized in that the erythropoietin content ranges from 100 IU / ml to 120,000 IU / ml.